Laser Desorption/Ionization (LDI) is an ionization technique in which a sample is irradiated with a laser light to desorb a substance and to help the change transfer to the substance. Matrix Assisted Laser Desorption/Ionization (MALDI) is another ionization technique suitable for ionizing proteins or other samples that hardly absorb the laser light or are easily damaged by the laser light. In MALDI, a substance that is likely to absorb the laser light and turn into ions is mixed into the sample beforehand as a matrix, and then the mixture is irradiated with a laser light to ionize the sample. Particularly, in recent years, mass spectrometers employing MALDI are widely used in life science or other fields because they enable the analysis of macromolecular compounds having large molecular weights without excessively dissociating the compounds. Moreover, they are also suitable for microanalysis. It should be noted that, in the present specification, mass spectrometers having an ion source using the LDI or MALDI method are generally referred to as the “LDI/MALDI-MS” system.
Microscopic mass spectrometers and imaging mass spectrometers are designed on different conceptual bases. Microscopic mass spectrometers are designed to perform a mass analysis using a visual image of the sample obtained through an optical observation; a microscopic image of the sample is observed through an optical microscope, the target position of the sample is specified on the observed image, and the mass analysis is carried out for the specified position. Imaging Mass spectrometry, on the other hand, are designed to create a fine two-dimensional image of the sample from signals obtained through a mass analysis; they use the result of the mass analysis to identify the texture of the microscopic image.
In any case, LDI/MALDI-MS systems can perform a mass analysis on a minute portion of the sample or obtain a mass image with high resolution by using a laser beam having a very small spot size (see Non-Patent Document 1 or Patent Document 1).
In the present application, these types of mass spectrometers are generally referred to as the “microscopic mass spectrometers.”
FIG. 1 shows an example of conventional microscopic mass spectrometers. The operator observes the sample 12 through the charge coupled device (CCD) 11 or ocular lens and specifies the target portion on the observed image. Subsequently, when he or she commands the system to start the analysis, the laser light source 13 casts a train of laser pulses onto the target portion of the sample 12. The observation optics and the laser-irradiation optics are appropriately located taking into account the above-described operations.
The analysis can be performed in various manners. For example, it is possible to specify one point at the time of observation and then carry out the mass analysis for only that point. Otherwise, one may specify a certain area (single or multiple areas) at the time of observation and carry out a two dimensional mass analysis for each area by scanning the area with the beam of laser light at the time of analysis. It is also possible to move the irradiation spot of the laser light beam along a straight or curved line to obtain a line profile of the sample.
The sample ionizes at the portion irradiated with the laser light, the generated ions 14 are pulled by the ion guide 15 into the mass analysis section 16, which performs the mass analysis of the ions. Thus, a mass spectrometry profile of the portion irradiated with the laser light is obtained.
The system shown in FIG. 1 includes an optical system for users to observe an accurate position of the target portion on the sample 12. In general, however, the microscopic mass analysis does not always require an elaborate optical observation system. For example, the microscopic mass analysis may take the following steps: the operator checks the position of the irradiation spot of the laser light by sight or through a simple optical observation means, after which the system performs the mass analysis while moving the sample stage or the irradiation spot of the laser light to obtain two-dimensional mass spectrometry information.
If the mass analysis requires a high level of mass resolution, it is advantageous to use a time-of-flight mass spectrometer (TOFMS) in the mass analysis section 16. The analysis using a TOFMS is based on the idea that the period of time required for an ion accelerated by an electric field to fly over a specific distance depends on the mass of the ion. That is, the period of time is measured from the time the ions are simultaneously released from a predetermined position to the time each ion is detected by the detector after it has flown through a space having a predetermined length. Although the laser light cast onto the sample is in the form of a very short pulse, it produces a large number of ions to be released from different positions with various initial velocities. When a sample is ionized under the atmospheric pressure, the variation on the time of flight of the ions is very large, so that a precise TOF analysis is difficult. To address these problems, an orthogonal acceleration TOFMS as shown in FIG. 1 has been used thus far. In this type of TOFMS, an acceleration voltage is applied in the direction orthogonal to the flying direction of the generated ions 14 so that the ions start their flight from approximately the same position with respect to the detector 17. The TOFMS shown in FIG. 1 is a reflectron type TOFMS, which may be replaced by a linear type TOFMS.
[Patent Document 1] U.S. Pat. No. 5,808,300
[Patent Document 2] Japanese Unexamined Patent Publication No. 2003-512702
[Non-Patent Document 1] Yasuhide NAITO, “Seitai Shiryou Wo Taishou Ni Shita Shituryou Kenbikyou (Mass Microprobe Aimed at Biological Samples)”, J. Mass Spectrom. Soc. Jpn., Vol. 53, No. 3, 2005, pp. 125-132
[Non-Patent Document 2] Michisato TOYODA, “Multi-turn Time-Of-Flight Mass Spectrometer ‘MULTUM Linear plus’ No Kaihatsu (Development of Multi-turn Time-Of-Flight Mass Spectrometer ‘MULTUM Linear plus’)”, J. Mass Spectrom. Soc. Jpn., Vol. 48, No. 5, 2000, pp. 312-317
One of the major objectives of the imaging mass spectrometry or the microscopic mass analysis is to analyze components of living tissue or living cells. In particular, analysis of proteins or sugars (saccharides) contained in a sample taken from a living body is in great demand. One of the effective methods for analyzing proteins, sugars or similar molecules is the MS/MS analysis, in which the ionized sample is dissociated by collision induced dissociation (CID) or similar methods to generate fragment ions (daughter ions), which are then fed to the analysis section. Use of an ion trap will significantly improve the efficiency of producing the fragment ions. The ion trap enables not only the simple MS/MS analysis but also the MSn analysis, in which the dissociation process repeatedly takes place.
The ion trap has a mass-analyzing capability by itself. However, it has only a low level of mass resolution if it is used independently. To solve this problem, it is advantageous to dispose a TOFMS 22 behind the ion trap 21, as shown in FIG. 2, in order to perform the mass analysis with high resolution during the MS/MS (or MSn) analysis. As shown in FIG. 3, the ion trap 21 temporarily stores ions within its inner space by the radio frequency (RF) voltage applied to the ring electrode 211 and then simultaneously ejects them outside when a direct voltage is applied to the two end cap electrodes 212, 213. The timing of the ejection can be synchronized with the timing at which the ions start their flight inside the TOFMS 22, whereby a high resolution of mass spectrum is obtained. This technique can be also applied to normal modes of MS analysis as well as the MSn analysis.
The combination of the ion trap 21 and the TOFMS 22 enables the MSn analysis to be efficiently performed and both the normal MS analysis and the MSn analysis to be carried out with high resolution. A laser mass spectrometer including an ion trap combined with a TOFMS as shown in FIG. 2 has already been realized. However, it does not function as a microscopic mass spectrometer.
In such mass spectrometers conventionally used, the storage, ejection and other operations of ions within the ion trap are performed by varying the amplitude of the voltage applied to the ring electrode of the ion trap. This method needs a high level of RF voltage to the ring electrode if an ion having a large mass (or a large mass-to-charge ratio) is to be trapped. However, generation of a high RF voltage requires a large-size power supply. Furthermore, the problem of electric discharge needs to be addressed. Thus, the conventional mass spectrometers have the limitation that they cannot practically trap the ions having large mass to charge ratios.
As stated earlier, there is a growing demand for microscopic mass spectrometry or imaging mass spectrometry that is applicable to the mass analysis of bio-samples. In the case of measuring a bio-sample, it is necessary to set the sample as is on the sample stage throughout the analysis. This setting makes it difficult to reduce the molecular weight of the sample by, for example, digesting the sample with an enzyme. Therefore, it is strongly desired that samples having large mass to charge ratios be analyzed at the ion trap.
Conventional mass spectrometers also have a problem relating to the mass resolution in addition to the above-described problem that the ion trap can trap ions only within a limited mass range. The mass resolution of conventional linear TOFMS or reflectron TOFMS is approximately 10000, while there are many proteins and other molecules whose mass to charge ratio exceeds tens of thousands. Therefore, it is impossible to carry out a satisfactory analysis with the conventional mass spectrometers when a highly accurate mass analysis of components of living tissue or living cells is demanded.
The object of the present invention is therefore to provide a laser irradiation mass spectrometer capable of solving the problems described thus far, which is particularly suitable for analyzing bio-samples.